PROJECT SUMMARY/ABSTRACT Candida albicans is usually a harmless commensal organism, but it can opportunistically invade the bloodstream of immunocompromised individuals and quickly spread to multiple organs, giving rise to life- threatening fungal infections. The limited arsenal of antifungal drugs, the prevalence of drug-resistant Candida strains, and significant gaps in understanding how fungal cells adapt to become pathogenic are serious threats to human health. The research proposed here explores the innovative and unexplored concept that changes in nutrient metal availability over the course of infection affect drug efficacy in currently unpredictable ways. At the same time, fungal responses to drug stress influence how fungi remodel their metallobiology to resist drug action and favor virulence. This hypothesis is based on preliminary data collected in the applicant's laboratory showing that the amount of copper in the growth medium of Candida albicans dramatically modulates the potency and resistance of three different classes of antifungal agents: transition metal ionophores, azoles, and antimicrobial peptides. These studies align with the applicant's long-term goals to develop chemical tools to manipulate biological metal ion location, speciation, and reactivity for potential therapeutic benefit. The overall objective of the current application is to identify targets, mechanisms, and pathways that confer metal-adjusted responses in drug efficacy against fungal species relevant to human health. This objective will be met by using a powerful combination of chemical, genetic and proteomic approaches to address three specific aims: 1) Determine how metal availability in the cellular growth environment affects phenotypic outcomes of fungal pathogens treated with common azole drugs; 2) Determine how metal availability modulates the cellular response to antifungal drug stress; and 3) Identify molecular and biological determinants of metal-modulated candidacidal activity of the antifungal histatin peptides. These aims will take advantage of growth media rigorously controlled for metal concentrations to correlate drug susceptibility of Candida albicans with measurable outcomes in growth inhibition, morphology and total cellular metal content. The second and third aims use both targeted and unbiased functional genomic and metalloproteomic approaches to identify genes and metalloproteins that influence or are influenced by treatment with azoles or histatins under conditions of variable metal levels. The assembled collaborative team of world-leading experts in fungal pathogenesis and metallobiology coupled with compelling preliminary results demonstrate feasibility of these strategies by the applicant. The impact of these studies for understanding how metal status affects antifungal drug efficacy will inform new directions for overcoming drug resistance and developing new antimicrobial strategies that take into account complex metallobiology along the host-pathogen interface.